CD4+ T helper cells are not a homogeneous population but can be divided on the basis of cytokine secretion into at least two subsets termed T helper type 1 (Th1) and T helper type 2 (Th2) (see e.g., Mosmann, T. R. et al. (1986) J. Immunol. 136:2348-2357; Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Th1 cells secrete interleukin-2 (IL-2) and interferon-xcex3 (IFN-xcex3) while Th2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13). Both subsets produce cytokines such as tumor necrosis factor (TNF) and granulocyte/macrophage-colony stimulating factor (GM-CSF). In addition to their different pattern of cytokine expression, Th1 and Th2 cells are thought to have differing functional activities. For example, Thl cells are involved in inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in providing efficient xe2x80x9chelpxe2x80x9d to B lymphocytes and stimulating production of IgG1 and IgE antibodies.
There is now abundant evidence that the ratio of Th1 to Th2 cells is highly relevant to the outcome of a wide array of immunologically-mediated clinical diseases including autoimmune, allergic and infectious diseases. For example, in experimental leishmania infections in mice, animals that are resistant to infection mount predominantly a Th1 response, whereas animals that are susceptible to progressive infection mount predominantly a Th2 response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72; Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is observed coincident with the release of eggs into the tissues by female parasites and is associated with a worsening of the disease condition (Pearce, E. J., et al. (1991) J. Exp. Med 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270). Many human diseases, including chronic infections (such as with human immunodeficiency virus (HIV) and tuberculosis) and certain metastatic carcinomas, also are characterized by a Th1 to Th2 switch (see e.g., Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988) Science 239:617-623). Furthermore, certain autoimmune diseases have been shown to be associated with a predominant Th1 response. For example, patients with rheumatoid arthritis have predominantly Th1 cells in synovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimental autoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1 cells (Kuchroo, V. K., et al. (1993) J. Immunol. 151:4371-4381).
The ability to alter or manipulate ratios of Th1 and Th2 subsets requires an understanding of the mechanisms by which the differentiation of CD4 T helper precursor cells (Thp), which secrete only IL-2, choose to become Th1 or Th2 effector cells. It is clear that the cytokines themselves are potent Th cell inducers and form an autoregulatory loop (see e.g., Paul, W. E. and Seder, R. A. (1994) Cell 76:241-251; Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673). Thus, IL-4 promotes the differentiation of Th2 cells while preventing the differentiation of precursors into Th1 cells, while IL-12 and IFN-xcex3 have the opposite effect. One possible means therefore to alter Th1:Th2 ratios is to increase or decrease the level of selected cytokines. Direct administration of cytokines or antibodies to cytokines has been shown to have an effect on certain diseases mediated by either Th1 or Th2 cells. For example, administration of recombinant IL-4 or antibodies to IL-12 ameliorate EAE, a Th1-driven autoimmune disease (see Racke; M. K. et al. (1994) J. Exp. Med 180:1961-1966; and Leonard, J. P. et al. (1995) J. Exp. Med. 181:381-386), while anti-IL-4 antibodies cure the Th2-mediated parasitic disease, Leishmania major (Sadick, M. D. et al. (1990) J. Exp. Med. 171:115-127). However, as therapeutic options, systemic administration of cytokines or antibodies may have unwanted side effects and, accordingly, alternative approaches to manipulating Th1/Th2 subsets are still needed.
The molecular basis for the tissue-specific expression of IL-4 in Th2 cells, or any T cell cytokine, has remained elusive. One possibility is the presence of repressor proteins that selectively silence cytokines. Transcriptional silencing has been well documented for bacteria, yeast and mammalian genes. Examples include E. coli thermoregulation genes (Goransson, M. et al. (1990) Nature 344:682-685), yeast xcex12 mating type genes (Keleher, C. A. et al. (1988) Cell 53:927-936) and mammalian MHC class I and TcRxcex1 genes (Weisman, J. D. and Singer, D. S. (1991) Mol. Cell. Biol. 11:4228-4234; Winoto, A. and Baltimore, D. (1989) Cell 59:649-655). Indeed, early experiments involving injection of IL-2 genomic DNA into Xenopus oocytes suggested the existence of a repressor protein for IL-2 in resting versus activated T cell extracts (Mouzaki, A. et al. (1991) EMBO J. 10:1399-1406). These studies suggested that the absence of IL-2 production in resting T cells was due to proteins that silenced the transcription of IL-2 by interacting with negative elements in the IL-2 promoter.
A second possibility is the existence of Th selective transactivators. A family of four related transcription factors called Nuclear Factor of Activated T cells (NF-AT), plays a key role in the regulation of cytokine gene expression (see e.g., Emmel, E. A. et al. (1989) Science 246:1617-1620; Flanagan, W. M. et al. (1991) Nature 352:803-807; Jain, J. et al. (1993) Nature 365:352-355; McCaffrey, P. G. et al. (1993) Science 262:750-754; Rao, A. (1994) Immunol. Today 15:274-281; Northrop, J. P. et al. (1994) Nature 369:497). However, NF-AT family members can bind to and transactivate the promoters of multiple cytokine genes including IL-2 and IL-4 (Rooney, J. et al. (1995) Immunity 2:545-553; Szabo, S. J. et al. (1993) Mol. Cell. Biol. 13:4793-4805; Flanagan, W. M. et al. (1991) Nature 352:803-807; Northrop, J. P. et al. (1994) Nature 369:497). Thus, they are not likely to be responsible for directing Th1- or Th2-specific cytokine transcription. Most, if not all, NF-AT binding sites in cytokine promoter regulatory regions are accompanied by nearby sites that bind auxiliary transcription factors, usually members of the AP-1 family. It has been shown that NF-AT and AP-1 proteins bind coordinately and cooperatively and are required for full activity of the IL-2 and IL-4 promoters. Different AP-1 proteins, specifically c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D, have been shown to bind to these sites (Rao, A. et al. (1994) Immunol. Today 15:274-281; Jain, J. et al. (1993) Nature 365:352-355; Boise, L. H. et al. (1993) Mol. Cell. Biol. 13:1911-1919; Rooney, J. et al. (1995) Immunity 2:545-553; Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). However, none of these AP-1 proteins is expressed in a Th1- or Th2-specific manner and there is no evidence for the differential recruitment of AP-1 family members to the IL-2 or IL-4 composite sites (Rooney, J. et al. (1995) Mol. Cell. Biol. 15:6299-6310). Thus, neither NF-AT proteins nor the AP-1 family members c-Jun, c-Fos, Fra-1, Fra-2, Jun B and Jun D can account for the tissue-specific transcription of IL-4 in Th2 cells.
This invention pertains to methods for regulating production of Th2-associated cytokines and for regulating Th1 or Th2 subsets by modulating the activity of one or more transcription factors that regulate expression of Th2-specific cytokine genes. As described further herein, it has now been discovered that the tissue-specific expression of IL-4 in Th2 cells is not due to a repressor protein but rather to a Th2-specific transactivator protein. The proto-oncogene c-Maf has now been demonstrated to be responsible for the tissue-specific expression of the Th2-associated cytokine interleukin-4. Moreover, ectopic expression of c-Maf in cells other than Th2 cells (e.g., Th1 cells, B cells and non-lymphoid cells) leads to activation of the IL-4 promoter and, under appropriate conditions, production of endogenous IL-4. It further has been discovered that c-Maf and NF-AT synergize to activate Th2-associated cytokine gene expression. It still further has been discovered that a third protein that interacts with NF-AT, termed NIP45 (for NF-AT Interacting Protein 45), potentiates gene expression mediated by c-Maf and NF-AT such that when all three factors (c-Maf, NF-AT and NIP45) are active in a cell, high levels of endogenous IL-4 production is stimulated. It still further has been discovered that a small maf protein lacking a transactivation domain, such as p18, can repress Th2-associated cytokine gene expression, e.g., expression mediated by c-Maf.
Accordingly, this invention pertains to methods for modulating Th2-associated cytokines expression by modulating the expression or activity of one or more transcription factors that cooperate with an NF-AT family protein to regulate the expression of Th2-associated cytokine genes. In one embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a Th2-specific transcription factor (e.g., a Th2-specific maf family protein). In one embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a maf family protein, such as c-Maf. In yet another embodiment, the transcription factor that cooperates with an NF-AT family protein to regulate the expression of a Th2-associated cytokine gene, and thus whose expression or activity is modulated, is a protein that interacts with an NF-AT family protein, such as NIP45. In yet another embodiment, the expression or activity of a small maf protein, such as p18, is modulated. The methods of the invention may involve modulating the expression or activity of one transcription factor (e.g., c-Maf or NIP45 or p18) or a combination of transcription factors (e.g., c-Maf+NF-AT, or NF-AT+NIP45, or c-Maf+NF-AT+NIP45).
The modulatory methods of the invention generally involve contacting a cell with an agent that modulates the expression or activity of a transcription factor(s) such that production of the Th2-associated cytokine by a cell is modulated. In particular, preferred agents of the invention act intracellularly to modulate the activity of the transcription factor. In one embodiment, the modulatory method of the invention stimulates production of a Th2-associated cytokine. For example, Th2-associated cytokine production can be stimulated in Th1 cells, B cells or non-lymphoid cells. In another embodiment, the modulatory method of the invention inhibits production of a Th2-associated cytokine. A Th2-associated cytokine modulated in the method preferably is interleukin-4.
A variety of agents can be used to stimulate the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. For example, a stimulatory agent of the invention can be a nucleic acid molecule encoding the transcription factor that is introduced into and expressed in the cell. Alternatively, chemical agents that enhance the expression or activity of the transcription factor can be used as stimulatory agents.
A variety of agents can be used to inhibit the expression or activity of a transcription factor that regulates expression of a Th2-associated cytokine gene. Examples of suitable inhibitory agents include antisense nucleic acid molecules that are complementary to a gene encoding the transcription factor, intracellular antibodies that bind the transcription factor (e.g., in the cell nucleus), inhibitory forms of the transcription factor (e.g., dominant negative forms) and chemical agents that inhibit the expression or activity of the transcription factor.
Combination methods, involving modulation of the expression or activity of two, three or more transcription factors that regulate Th2-associated cytokine gene expression, are also encompassed by the invention. Accordingly, in other embodiments of the invention, a cell is contacted with at least one additional agent that modulates the activity of at least one additional transcription factor that contributes to the regulation of the Th2-associated cytokine gene. Preferably, the at least one additional transcription factor whose expression or activity is modulated is selected from the group consisting of NF-AT family proteins, NF-AT-interacting proteins, maf family proteins and AP-1 family proteins.
Cytokine production by a cell can be modulated in vitro or in vivo in accordance with the methods of the invention. In one embodiment, a cell is contacted with a modulating agent(s) in vitro and then the cell is administered to a subject to thereby regulate the development of Th1 and/or Th2 subsets in the subject. Accordingly, in another aspect, the invention provides methods for regulating the development of Th1 or Th2 subsets in a subject. In addition to the embodiment wherein ex vivo modified cells are administered to the subject, in another embodiment, these methods involve direct administration to the subject of an agent that modulates the activity of one or more transcription factors that regulate expression of a Th2-associated cytokine gene such that development of Th1 or Th2 cells in the subject is modulated.
The modulatory methods of the invention can be used to manipulate Th1:Th2 ratios in a variety of clinical situations. For example, inhibition of Th2 formation may be useful in allergic diseases, malignancies and infectious diseases whereas enhancement of Th2 formation may be useful in autoimmune diseases and organ transplantation.